A systematic study of Tupaia as a model for human acute hepatitis B infection.

The molecular features of hepatitis B virus (HBV) infection, eradication, and pathogenesis are poorly understood, partly due to the lack of an adequate animal model that faithfully reproduces the course of infection. Although Tupaia belangeri were previously recognized as HBV-susceptible animals, the course of infection in adult tupaias remains obscure. Herein, we performed a longitudinal study and demonstrated that adult tupaias were efficiently infected (90% infection rate) with 108 copies of the HBV genome. HBV replicated vigorously, produced high levels of covalently closed circular DNA (cccDNA) in hepatocytes, and released hepatitis B surface antigen (HBsAg), hepatitis Be antigen (HBeAg), and HBV DNA into the serum at day 9 post-inoculation (p.i.), which then decreased on day 15 p.i. The kinetics were consistent with the expression of liver HBsAg and HBeAg, as determined with immunohistochemistry. The viral products in serum at day 9 and 15 p.i. represented de novo synthesized viral products, as treatment with a viral entry inhibitor completely abolished these products from the serum. Viral clearance and serological conversion occurred at day 21 p.i. and were accompanied by elevated alanine transaminase (ALT) levels and liver pathology, such as inflammatory infiltration and hepatocyte ballooning degeneration. Although ALT levels eventually returned to normal levels by day 42 p.i., the liver pathology persisted until at least day 120 p.i. The HBV infection process in tupaia, therefore, exhibits features similar to that of human acute HBV infection, including viral replication, viral eradication, ALT elevation, and liver pathology. Thus, adopting the tupaia model to study host-HBV interactions presents an important advance which could facilitate further investigation and understanding of human HBV infection, especially for features like cccDNA that current small-animal models cannot effectively model.

Effect of haptoglobin on the treatment of chronic hepatitis B with interferon.

There are two main types of drugs that are used to treat chronic hepatitis B (CHB), including interferon (IFN) and nucleotide analogues. IFN inhibits the virus through direct antiviral activity and via immune regulation, and it has been widely applied for the treatment of CHB and other infections. However, the efficiency of IFN therapy is not entirely satisfactory. The aim of the present study was to investigate the factors affecting IFN therapy. The plasma of patients with CHB treated with IFN was collected and divided into the virological response group and non-virological response group according to their virological response. Serum proteins of virologically responsive patients were compared before and after IFN therapy using isobaric tags for relative and absolute quantitation technology. ELISA was used to validate these results in the same sample. In in vitro cell experiments, HepG2.2.15 cells were transfected with haptoglobin (Hp)-targeting small interfering RNA to inhibit expression of the Hp protein, and reverse transcription-quantitative polymerase chain reaction and western blotting were utilized to detect hepatitis B virus (HBV)-DNA, IFN and downstream molecular changes in the cell supernatants. The Hp protein levels were demonstrated to be significantly lower following 48 weeks of IFN therapy, and the levels of Hp in patients in the virological response group were significantly lower than those in the non-virological response group. In in vitro cell experiments, following inhibition of Hp protein expression, significantly decreased levels of HBV-DNA, and elevated levels of IFN and its downstream molecules were observed. These findings suggest that Hp may be able to predict the efficacy of IFN therapy, and it may inhibit HBV clearance. There is an association between Hp and IFN, which requires further clinical and laboratory studies to explore.

Relationship between hepatitis B virus infection and chronic kidney disease in Asian populations: a meta-analysis.

PURPOSE: To evaluate the association of Chronic hepatitis B virus (HBV) infection and chronic kidney disease (CKD). METHODS: We searched Embase, Grateful Med, Ovid, PubMed, and the China Biological Medicine Database. A meta-analysis was performed to assess whether HBV infection plays an independent impact on the development of CKD in the general population. Relative risks of CKD (defined as reduced glomerular filtration rate or proteinuria) according to HBsAg serologic status were studied. RESULTS: Six eligible clinical studies (189,709 individuals in total) were included in the analysis. There was no association between HBsAg seropositive status and prevalence of CKD, the summary estimate for adjusted relative risk (RR) was 1.16 (95% confidence interval (CI), 0.78, 1.71; p = .46) according to the random-effects model, and between studies heterogeneity was noted (p values by Q test <0.001). Also, there were no significant associations between positive HBV serologic status and low eGFR (adjusted relative risk, 0.95; 95% CI, 0.72, 1.26; p = .72) or proteinuria (adjusted relative risk, 1.00; 95% CI, 0.83, 1.20; p = .99). CONCLUSIONS: This meta-analysis shows that there was no association between exposure to HBV and the risk of developing CKD in Asian populations.

[Treatment with interferon and thymosin alpha-1 versus interferon monotherapy for HBeAg positive chronic hepatitis B: a meta-analysis].

To compare the efficacy of interferon and thymosin alpha-1 combination therapy with interferon monotherapy for HBeAg positive chronic hepatitis B. The relevant randomized controlled trials were searched throughout PubMed, EBSCO, Cochrane Library, CBMdisc, VIP, WanFang since Janurary 1990. Studies were included if patients were followed up for at least 6 months after cessation of treatment. Meta-analysis was carried out with RevMan5.0 software. Subgroup analyses were used at different time of observation. Seven randomized controlled trials were included(535 patients in total). According to the results of meta-analysis, the combination therapy was remarkably more effective than monotherapy both at the end of the treatment and the follow-up in terms of HBV-DNA negative rate (54.9% vs 36.3%, OR=2.39, 95% CI=1.64-3.49, P value is less than 0.01; 58.6% vs 30.7%, OR=3.68, 95% CI=2.51-5.41, P value is less than 0.01, respectively), ALT normalization rate (74.5% vs 60.9%, OR=1.94, 95% CI=1.26-3.00, P value is less than 0.01; 74.0% vs 55.6%, OR=2.36, 95% CI=1.54-3.62, P value is less than 0.01, respectively), HBeAg loss rate (56.9% vs 36.7%, OR=2.38, 95% CI=1.61-3.51, P value is less than 0.01; 62.2% vs 33.2%, OR=3.42, 95% CI=2.31-5.06, P value is less than 0.01, respectively) , and HBeAg seroconversion rate (40.1% vs 29.0%, OR=1.65, 95% CI=1.10-2.47, P value is less than 0.05; 47.0% vs 29.5%, OR=2.13, 95% CI=1.43-3.16, P value is less than 0.01, respectively); the HBsAg loss rate of the combination therapy group was significantly higher than that of the monotherapy group only at the end of the follow-up (9.8% vs 3.7%, OR=2.92, 95% CI=1.09-7.76, P value is less than 0.05). Interferon and thymosin alpha-1 combination therapy achieves superior effect with no increase in the adverse effects as compared to interferon monotherapy for HBeAg positive chronic hepatitis B.

Therapeutic polypeptides based on HBV core 18-27 epitope can induce CD8+ CTL-mediated cytotoxicity in HLA-A2+ human PBMCs.

AIM: To explore how to improve the immunogenicity of HBcAg CTL epitope based polypeptides and to trigger an HBV-specific HLA I-restricted CD8+ T cell response in vitro. METHODS: A new panel of mimetic therapeutic peptides based on the immunodominant B cell epitope of HBV PreS2 18-24 region, the CTL epitope of HBcAg18-27 and the universal T helper epitope of tetanus toxoid (TT) 830-843 was designed using computerized molecular design method and synthesized by Merrifield's solid-phase peptide synthesis. Their immunological properties of stimulating activation and proliferation of lymphocytes, of inducing T( H1) polarization, CD8+ T cell magnification and HBV-specific CD8+ CTL mediated cytotoxicity were investigated in vitro using HLA-A2+ human peripheral blood mononuclear cells (PBMCs) from healthy donors and chronic hepatitis B patients. RESULTS: Results demonstrated that the therapeutic polypeptides based on immunodominant HBcAg18-27 CTL, PreS2 B- and universal T(H) epitopes could stimulate the activation and proliferation of lymphocytes, induce specifically and effectively CD8+ T cell expansion and vigorous HBV-specific CTL-mediated cytotoxicity in human PBMCs. CONCLUSION: It indicated that the introduction of immunodominant T helper plus B-epitopes with short and flexible linkers could dramatically improve the immunogenicity of short CTL epitopes in vitro.

Therapeutic polypeptides based on HBcAg(18-27) CTL epitope can induce antigen-specific CD(8)(+) CTL-mediated cytotoxicity in HLA-A2 transgenic mice.

AIM: To explore how to trigger an HLAI-restricted CD8(+) T cell response to exogenously synthesized polypeptides in vivo. METHODS: Three mimetic therapeutic polypeptides based on the immunodominant CTL epitope of HBcAg, the B- epitope of HBV PreS(2) region and a common T helper sequence of tetanus toxoid were designed and synthesized with Merrifield's solid-phase peptide synthesis method. Their immunological properties of inducing T( H1) polarization, CD8(+) HBV-specific CTL expansion and CD8(+) T cell mediated cytotoxicity were investigated in HLA-A2 transgenic mice. RESULTS: Results demonstrated that the mimetic polypeptides comprised of the immunodominant CTL, B-, and T helper epitopes could trigger specifically and effectively vigorous CD8(+) HBV-specific CTL-mediated cytotoxicity and T(H1) polarization of T cells in HLA-A2 transgenic mice. CONCLUSION: A designed universal T helper plus B-epitopes with short and flexible linkers could dramatically improve the immunogenicity of CTL epitopes in vivo. And that the mimetic therapeutic peptides based on the reasonable match of the above CTL, B- and T helper epitopes could be a promising therapeutic peptide vaccine candidate against HBV infection.

Construction and characterization of an experimental ISCOMS-based hepatitis B polypeptide vaccine.

AIM: To characterize the biochemical and immunological properties of an experimental ISCOMS vaccine prepared from a novel therapeutic polypeptide based on T cell epitopes of HBsAg, and a heptatis B-ISCOMS was prepared and investigated. METHODS: An immunostimulating complexes(ISCOMS)-based vaccine containing a novel therapeutic hepatitis B polypeptide was prepared by dialysis method, and its formation was visualized by electron microscopy and biochemically verified by SDS-polyacrylamide gel electrophoresis. Amount of the peptide within ISCOMS was determined by Bradford assay, and specific CTL response was detected by ELISPOT assay. RESULTS: Typical cage-like structures of submicroparticle with a diameter of about 40nm were observed by electron microscopy. Results from Bradford assay showed that the level of peptide incorporation was about 0.33g.L(-1). At the paralleled position close to the sixth band of the molecular weight marker(3480kDa) a clear band was shown in SDS-PAGE analysis, indicating successful incorporation of polypeptide into ISCOMS. It is suggested that ISCOMS delivery system could efficiently improve the immunogenicity of polypeptide and elicit specific immune responses in vivo by the results of ELISPOT assay, which showed that IFN-gamma producing cells(specific CTL responses) were increased(spots of ISCOMS-treated group: 47+/-5, n =3; control group: 5+/-2, n =3). CONCLUSION: ISCOMS-based hepatitis B polypeptide vaccine is successfully constructed and it induces a higher CTL response compared with short polypeptides vaccine in vivo.